ABSTRACTTo understand colon cancer metastasis, we earlier analyzed a mouse model that developed liver metastasis of cancer cells disseminated from the spleen. We suggested that CCR1(+) bone marrow (BM)-derived cells are recruited to the microenvironment of disseminated colon cancer cells, and produce metalloproteinases MMP9 and MMP2, helping metastatic colonization. In the present study, we have examined these myeloid cells expressing CCR1 and/or MMPs in detail. To this end, we have established bacterial artificial chromosome (BAC)-based transgenic mouse lines in which membrane-targeted Venus fluorescent protein (mVenus) was expressed under the control of Ccr1 gene promoter. Then, myeloid cells obtained from the BM and liver metastatic foci were analyzed by the combination of flow cytometry and cytology/immunohistochemistry, in situ RNA hybridization, or quantitative RT-PCR. We have found four distinct types of myeloid cells recruited to the metastatic foci; neutrophils, eosinophils, monocytes and fibrocytes. These cell types exhibited distinct expression patterns for CCR1, MMP2 and MMP9. Namely, neutrophils found in the early phase of cancer cell dissemination expressed CCR1 exclusively and MMP9 preferentially, whereas fibrocytes accumulated in later phase expressed MMP2 exclusively. Either genetic inactivation of Ccr1 or antibody-mediated neutrophil depletion reduced subsequent recruitment of fibrocytes. The recruitment of CCR1(+) neutrophils in early phase of colon cancer dissemination appears to cause that of fibrocytes in late phase. These results implicate the key role of CCR1 in colon cancer metastasis in this mouse model, and explain why both MMP9 and MMP2 are essential as genetically demonstrated previously. The results also suggest relevant mechanisms in humans.

Fig2: Most CCR1+ cells accumulating in the liver metastatic foci are neutrophils.(a and b) Immunofluorescence analysis of the liver metastatic foci on day 14 post-transplantation. Expression of CD45 (a pan-leukocyte marker) and Ccr1-mVenus reporter are shown for small (upper photos) and large (lower photos) metastatic liver foci (a). A large metastatic liver focus analyzed for expression of Ccr1-mVenus and Gr-1 (a mouse neutrophil marker) (b). Nuclei were stained with DAPI. Scale bar, 100 μm. T: tumor, L: liver tissue. c Chronological changes in the proportion of CD45+ hematopoietic cells accumulated in the metastatic foci of the liver. Cells harvested from the liver at the indicated days were analyzed by flow cytometry. Propidium iodide (PI)− CD45+ live hematopoietic cells circled in the upper panels were further analyzed for expression of Ccr1-mVenus and Gr-1 in the lower panels. Cells outside the circles were hepatocytes, resident stromal cells and/or debris, and therefore, excluded from further analyses. Numbers in the panels show the percentages of CD45+ cells (top) or Ccr1-mVenus+ cells within the CD45+ cells (bottom). d Most Ccr1-mVenus+ cells in the liver at day 14 after tumor transplantation were CD11b+ Gr-1+ (left and center), and showed the morphology characteristic of mature neutrophils (right). Scale bar, 10 μm

Mentions:
As we reported previously [16], intrasplenic injection of CCL9-expressing CMT93 colon cancer cells can cause their dissemination into the liver, which is followed by metastatic colonization. Employing this model, we monitored the behavior of the Ccr1-mVenus+ cells in the cancer metastasis microenvironment first by histochemistry at 14 days post-injection. We found that the density of Ccr1-mVenus+ cells among CD45+ hematopoietic cells was relatively high (~50/0.01 mm2) in small foci (i.e., Φ < 0.2 mm; Fig. 2a, top) whereas it was very low (<5/0.01 mm2) in large foci (Φ > 0.2 mm; Fig. 2a, bottom). These results suggest that accumulation of CCR1+ cells at the metastatic foci takes place at earlier stages around micrometastases rather than at later stages of colonization. At a higher magnification, 50–80 % of the Ccr1-mVenus+ cells were Gr-1+ neutrophils, whereas the remaining Gr-1− cells contained both mononuclear and non-mononuclear cells (Fig. 2b).Fig. 2

Fig2: Most CCR1+ cells accumulating in the liver metastatic foci are neutrophils.(a and b) Immunofluorescence analysis of the liver metastatic foci on day 14 post-transplantation. Expression of CD45 (a pan-leukocyte marker) and Ccr1-mVenus reporter are shown for small (upper photos) and large (lower photos) metastatic liver foci (a). A large metastatic liver focus analyzed for expression of Ccr1-mVenus and Gr-1 (a mouse neutrophil marker) (b). Nuclei were stained with DAPI. Scale bar, 100 μm. T: tumor, L: liver tissue. c Chronological changes in the proportion of CD45+ hematopoietic cells accumulated in the metastatic foci of the liver. Cells harvested from the liver at the indicated days were analyzed by flow cytometry. Propidium iodide (PI)− CD45+ live hematopoietic cells circled in the upper panels were further analyzed for expression of Ccr1-mVenus and Gr-1 in the lower panels. Cells outside the circles were hepatocytes, resident stromal cells and/or debris, and therefore, excluded from further analyses. Numbers in the panels show the percentages of CD45+ cells (top) or Ccr1-mVenus+ cells within the CD45+ cells (bottom). d Most Ccr1-mVenus+ cells in the liver at day 14 after tumor transplantation were CD11b+ Gr-1+ (left and center), and showed the morphology characteristic of mature neutrophils (right). Scale bar, 10 μm

Mentions:
As we reported previously [16], intrasplenic injection of CCL9-expressing CMT93 colon cancer cells can cause their dissemination into the liver, which is followed by metastatic colonization. Employing this model, we monitored the behavior of the Ccr1-mVenus+ cells in the cancer metastasis microenvironment first by histochemistry at 14 days post-injection. We found that the density of Ccr1-mVenus+ cells among CD45+ hematopoietic cells was relatively high (~50/0.01 mm2) in small foci (i.e., Φ < 0.2 mm; Fig. 2a, top) whereas it was very low (<5/0.01 mm2) in large foci (Φ > 0.2 mm; Fig. 2a, bottom). These results suggest that accumulation of CCR1+ cells at the metastatic foci takes place at earlier stages around micrometastases rather than at later stages of colonization. At a higher magnification, 50–80 % of the Ccr1-mVenus+ cells were Gr-1+ neutrophils, whereas the remaining Gr-1− cells contained both mononuclear and non-mononuclear cells (Fig. 2b).Fig. 2

Bottom Line:
We have found four distinct types of myeloid cells recruited to the metastatic foci; neutrophils, eosinophils, monocytes and fibrocytes.Either genetic inactivation of Ccr1 or antibody-mediated neutrophil depletion reduced subsequent recruitment of fibrocytes.The results also suggest relevant mechanisms in humans.

ABSTRACTTo understand colon cancer metastasis, we earlier analyzed a mouse model that developed liver metastasis of cancer cells disseminated from the spleen. We suggested that CCR1(+) bone marrow (BM)-derived cells are recruited to the microenvironment of disseminated colon cancer cells, and produce metalloproteinases MMP9 and MMP2, helping metastatic colonization. In the present study, we have examined these myeloid cells expressing CCR1 and/or MMPs in detail. To this end, we have established bacterial artificial chromosome (BAC)-based transgenic mouse lines in which membrane-targeted Venus fluorescent protein (mVenus) was expressed under the control of Ccr1 gene promoter. Then, myeloid cells obtained from the BM and liver metastatic foci were analyzed by the combination of flow cytometry and cytology/immunohistochemistry, in situ RNA hybridization, or quantitative RT-PCR. We have found four distinct types of myeloid cells recruited to the metastatic foci; neutrophils, eosinophils, monocytes and fibrocytes. These cell types exhibited distinct expression patterns for CCR1, MMP2 and MMP9. Namely, neutrophils found in the early phase of cancer cell dissemination expressed CCR1 exclusively and MMP9 preferentially, whereas fibrocytes accumulated in later phase expressed MMP2 exclusively. Either genetic inactivation of Ccr1 or antibody-mediated neutrophil depletion reduced subsequent recruitment of fibrocytes. The recruitment of CCR1(+) neutrophils in early phase of colon cancer dissemination appears to cause that of fibrocytes in late phase. These results implicate the key role of CCR1 in colon cancer metastasis in this mouse model, and explain why both MMP9 and MMP2 are essential as genetically demonstrated previously. The results also suggest relevant mechanisms in humans.